Fibrotic processes are associated with a wide range of physiological disorders. Scarring can be problematic in nearly all organs and tissues of the body, e.g. eyes, lungs, central nervous system, muscle, joints, kidney etc. Similar processes may also result in other fibrotic disorders which are common in many areas of medicine and surgery. For example, abdominal surgery often leads to intraperitoneal fibrous adhesions and/or strictures, whilst fibrotic retinopathy, scarring following glaucoma surgery, fibrosis of the trabecular meshwork, proliferative vitreoretinopathy, keloids and hypertrophic scars, skin diseases (e.g. epidermolysis bullosa, scleroderma), systemic sclerosis, pulmonary fibrosis, glomerulo nephritis, tubule-interstitial kidney fibrosis, myocardial fibrosis after myocardial infarct, scarring of the central nervous system following, for example a stroke or neurosurgery and hepatic cirrhosis are significant medical problems. Inappropriate scarring can compromise joint function after tendon damage.
Adult wound healing is characterised by acute inflammation, contraction and collagen deposition, responses likely to have been optimised for rapid wound closure and minimising infection. In brief, when the human body or the body of any mammal is wounded, a set of complex biochemical events takes place to repair the damage. These events start with an inflammatory phase which leads to the release of cytokines that cause the migration and division of cells involved in the proliferative phase. At this stage collagen deposition, granulation tissue formation, epithelialization, and wound contraction are observed. Fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting collagen and fibronectin. Epithelial cells migrate and cover the wound, and then the wound is made smaller by the contraction of myofibroblasts. Finally in the remodeling phase, collagen is remodeled and realigned along tension lines and cells that are no longer needed are removed by apoptosis.
Inflammation and pro-fibrotic molecules induce the transformation of epithelial cells into mesenchymal cells (EMT), promoting the accumulation of fibroblasts and myofibroblasts, fibrosis and migration of activated mesenchymal cells to expand the fibrotic foci. This process of cellular transdifferentiation is largely regulated by transforming growth factor-beta (TGF-beta).
In most cases, wound healing leads to the formation of well constructed scar tissue. However, in cases when one or more steps in this cascade are not well regulated serious consequences to health and appearance may result. These may arise from aberrant deposition of collagen and fibronectin which is observed in scleroderma and idiopathic pulmonary fibrosis and many related disorders, for example. Furthermore, aberrant tissue repair may occur after surgical procedures, including eye surgery, abdominal surgery, spinal surgery and the repair of surface wounds leading to keloid formation. It may be necessary to attenuate normal tissue repair in some instances; for example after a surgical procedure to drain aqueous humor from the eye, the scarring process can prematurely close the small drainage hole. Systemic diseases including diabetes and hypertension could induce injury of the glomerular and tubular cells in the kidney resulting in kidney fibrosis and renal failure.
The scarring process can be understood from a biochemical perspective as a TGF-beta initiated cascade of events. TGF-beta derives from latent transforming growth factor-beta (LTGF-beta). Thus the activation of LTGF-beta to active TGF-beta is a critical step in the healing and fibrotic processes. LTGF-beta (which comprises TGF-beta bound to the latency associated peptide (LAP), which in turn may be bound to the LTGF-beta binding protein (LTBP)), binds to cell-surface cation-independent mannose-6-phosphate (CI-M6P) receptors through the recognition of M6P-modified amino acids in the LAP. This binding allows the activation of the LTGF-beta initiated by a cascade of events which ultimately lead to the release of TGF-beta.
Since LTGF-beta binds to the CI-M6P receptor, CI-M6P receptor antagonists can play a significant role in the healing process by competing with the M6P-containing carbohydrates in the LAP for the CI-M6P receptor binding site. By acting in this way phosphotetrahydropyran CI-M6P receptor antagonists can attenuate or completely inhibit the binding of LTGF-beta to the M6P receptor, and the levels of active TGF-beta will be reduced.
Connective tissue growth factor (CTGF) is a secreted cytokine believed to be a central mediator in these cellular processes. In particular, CTGF is known to increase extracellular matrix production via increased deposition of collagen and fibronectin. Over-expression of CTGF has been implicated as a major causative factor in conditions such as scleroderma, fibroproliferative diseases, and scarring in is which there is an over accumulation of extracellular matrix components.
It has been reported that transforming growth factor beta (TGF-beta) and connective tissue growth factor (CTGF) are over-expressed in a coordinated fashion in an in vivo model of wound repair, suggesting that
TGF-beta-stimulated CTGF expression is involved in the healing of wounds. In a mouse model of skin fibrosis the subcutaneous injection of TGF-beta or CTGF had only minor effects when administered alone, but when together, a more persistent fibrotic response was observed. Therefore it is likely that CTGF acts as a downstream effector of TGF-beta acting to enhance the production of scar tissue. It has also been shown that inhibition of CTGF expression by antisense mRNA or by CTGF-binding antibodies prevents increased collagen synthesis in fibroblasts exposed to TGF-beta suggesting that CTGF induction is essential for the fibrotic response to TGF-beta. Suppression of CTGF might prevent a progressive fibrotic response to stimulation by TGF-beta.
Increased levels of TGF-beta, induced by inflammation, high glucose, hypoxia or other types of injury, are implicated in the modulation of a variety of cellular and molecular processes leading to cell activation, proliferation and migration. TGF-beta modulates molecular interactions with MAPK, PI3K/Akt, Wnt, Hedgehog, Notch and HIF pathways in a complex and context-dependent crosstalk cell signalling pathway.
Many of these fibrotic diseases and scarring disorders are very poorly treated with current medication, one example being idiopathic pulmonary fibrosis (IPF). IPF is a chronic lung disease of unknown origin characterised by chronic progressive interstitial lung fibrosis causing dyspnoea due to poor gas exchange and non-productive cough. Classically a disease of adulthood, IPF has a poor prognosis and no proven effective treatment. IPF is usually fatal with a life expectancy of three years after diagnosis, with cor pulmonale and cardiac failure being the main causes of death. Histopathological examination evidences alveolar damage with aberrant epithelial repair and interstitial pneumonia with overproduction of collagen, fibronectin and other ECM components by activated fibroblasts.
IPF development has been strongly correlated with cigarette smoking, and exposure to either silica or livestock. Other studies have suggested a correlation with viral infections. While the etiology of the disease remains to be understood, the pathogenesis seems to follow a model initiated by alveolar epithelial cell injury resulting in release of pro-inflammatory mediators as well as fibroblast proliferation similar to the process normally seen during tissue repair and would healing. In IPF patients, the lung injury cycle persists and the repair process never resolves, resulting in progressive fibrosis and loss of normal lung tissue architecture.
Acute inflammation of alveolar tissue has been proposed to be the first stage of IPF. This inflammatory process, started with an initial injury, is characterized by the acute recruitment of neutrophils and followed by migration of monocytes, lymphocytes and other immune-cells into the alveolar space. This induces epithelial and fibroblast activation accompanied by release of inflammatory mediators where TGF-beta, TNF and platelet derived growth factor play important roles. In susceptible individuals, acute inflammation does not resolve and gives rise instead to chronic inflammatory changes, abnormal alveolar tissue repair and remodeling which results in progressive fibrosis. Fibrogenic cytokines, including TGF-beta and TNF, induce myofibroblast migration and accumulation in the lung tissue, which in turn promote extracellular matrix deposition, collagen accumulation and other features of the fibrotic component of IPF.
There is an urgent need for medication to control both the initial inflammatory insult as well as the subsequent sequelae of fibrolytic and scarring events in patients suffering from IPF.
The eye is a very sensitive organ that is prone to injury and tissue tear and is frequently subjected to surgical procedures. In all instances aberrant wound repair can lead to ocular disorders; these are associated with inflammatory and cellular processes described previously. An over accumulation of extracellular matrix materials in the region of the trabecular meshwork (TM) is observed in forms of glaucoma; such increases are believed to lead to increased resistance to aqueous outflow and, therefore, elevated intraocular pressure (IOP).
The TM is a complex tissue including trabecular cells, connective tissue, and extracellular matrix located at the angle between the cornea and iris that provides the normal resistance required to maintain a normal IOP. An adequate IOP is needed to maintain the shape of the eye and to provide a pressure gradient to allow for the flow of aqueous humor to the avascular cornea and lens. Excessive IOP, commonly present in glaucoma, has deleterious effects on the optic nerve, leads to loss of retinal ganglion cells and axons, and results in progressive visual loss and blindness if not treated. Glaucoma is one of the leading causes of irreversible visual impairment and blindness worldwide.
Most forms of glaucoma result from disturbances in the flow of aqueous humor that have an anatomical, biochemical or physiological basis. Primary open angle glaucoma (POAG), also known as chronic or simple glaucoma, represents the majority of all glaucomas in the United States. POAG is characterized by pathological changes in the TM, resulting in abnormally high resistance to fluid drainage from the eye. A consequence of such resistance is an increase in the IOP.
Current anti-glaucoma therapies lower IOP by the use of medications to suppress aqueous humor formation or to enhance aqueous outflow. Unfortunately, the use of drug therapy alone is not sufficient to adequately control intraocular pressure in, some patients, particularly if there is a severe blockage of the normal passages for the outflow of aqueous humor. Such patients may require surgical intervention to restore the normal outflow of aqueous humor and thereby normalize or at least control their intraocular pressure. The outflow of aqueous humor can be improved by means of various intraocular surgical procedures, such as trabeculectomy, posterior lip sclerectomy, trephine and thermal sclerostomy. These surgical procedures are collectively referred to herein as glaucoma filtration surgery (GFS).
GFS, in particular a guarded sclerostomy procedure otherwise known as trabeculectomy, is a key point in the long term management of a patient's disease. Glaucoma surgery is traditionally performed on patients who have uncontrolled intraocular pressures (IOP) whilst on maximally tolerated medical treatment, or after failed laser trabeculoplasty and requires the surgeon to have great control over post-operative scarring to control the potential visual loss which can occur from glaucomatous damage if the intraocular pressures are not adequately controlled.
The procedures utilized in glaucoma filtration surgery generally involve the creation of a fistula to promote the drainage of aqueous humor. Although various procedures have been utilized, the procedures will typically include the creation of an elevation of the conjunctiva at the surgical site. This elevation is commonly referred to as the “filtering bleb”. The filtering blebs which are most often associated with good intraocular pressure control are avascular and either low and diffuse or elevated with numerous cystic spaces. Studies have suggested that aqueous fluid in the filtering bleb usually filters through the conjunctiva and mixes with the tear film, or is adsorbed by vascular or perivascular conjunctival tissue. Although glaucoma filtration surgery is generally successful initially, it is often plagued by the formation of scar tissue which may ultimately block the fistula created during the surgery. Increased amount of collagen in the failed fistulas suggests that proliferation of fibroblasts and associated production of extracellular matrix materials, particularly collagen, fibronectin and glycosaminoglycans were observed.
In order to prevent the scarring process the use of antimetabolite/antifibrotic drugs has emerged as an important adjunct to glaucoma filtration surgery. Among the different drugs tested the following are the ones most frequently used at present. 5-Fluorouracil (5-FU) is a pyrimidine analogue that acts by selectively inhibiting DNA synthesis in the S and the G2 phases of the cell cycle. Mitomycin C (MCC) acts by reducing fibroblast collagen synthesis through, inhibition of DNA-dependant RNA synthesis and has a direct cytotoxic effect. However, there are serious complications with current treatments including corneal epithelial toxicity, increased conjunctival wound leak frequency, hypotony and hypotony maculopathy, increased incidence of suprachordial hemorrhage, accurate dosimetry due to variability of delivery of drug between impregnated sponge and subconjunctival tissues, leakage away from treatment site leading to extra/intra ocular toxicity, and high levels of cell apoptosis.
Due to these limitations with current drug regimens, there is an urgent need for new methods to control both the initial inflammatory insult as well as the subsequent sequelae of scarring events to better preserve the filtering bleb.
Another unmet medical need is the control of excessive cutaneous scarring that causes functional, cosmetic and psychological morbidity. Clinical scar management after trauma, surgical injury or burns involves consideration of both the continual physical assessment of the scar, including bodylocation and the patient's previous scar history, with a clinical regimen that is often modulated over the course of treatment. Accepted conservative treatments for hypertrophic scars and keloids are limited to surgery, corticosteroid injections, radiotherapy, silicone gel sheeting and pressure therapy. Treatments that specifically target the biological mechanisms responsible for hypertrophic scars and keloids would complement existing therapy and could improve current scar outcome.
Cutaneous scarring, which is described as macroscopic disruptions of normal skin architecture and function, arises as a consequence of wound repair and proceeds as a fibroproliferative response. Keloids are hallmarked by growth beyond the margins of the original trauma site, are associated with familial disposition, and rarely regress. Hypertrophic scars are raised, erythematous fibrous lesions which usually undergo resolution over time and are associated with contracture of tissue.
In view of the serious consequences when the wound healing process goes awry, there is an urgent need for new medicines to control one or more steps of the cascade of biochemical events. Most appealing would be methods to modulate both the initial and ongoing inflammatory events and the subsequent collagen and fibronectin deposition. Where the human disease or disorder is well established, such methods are therapeutic leading to amelioration of disease symptoms. However, such methods can also be used prophylactically when administered at the time of surgical intervention and for defined periods following surgery.
Renal function impairment and progression to kidney failure are serious consequences of chronic systemic diseases including type 2 diabetes and hypertension. In type 2 diabetes, disruption of the glomerular filtering function and thickening of the tubular apparatus driven by high glucose levels in serum and tissue hypoxia lead to progressive renal function impairment and almost inevitable late stage renal failure driven by fibrotic tissue deposition in the renal parenchyma. Lack of effective therapeutic agents make this field an unmet medical need.